Bacterial toxins play major roles in the virulence of a large number of bacterial pathogens. They exert their toxic effects by modifying proteins that are integral to eukaryotic cell function. Although the effects of different bacterial toxins are diverse, a common ADP-ribosyltransferase molecular mechanism of action has been identified for many of them. Sequence comparisons within the enzymatically active region of these toxins, however, offer few clues as to how a common activity has been maintained. The major objective of studies described in this proposal is to determine what defines the common ADP-ribosyltransferase activity of these toxins. To extend an understanding of the ADP-ribosyltransferase reaction, the following specific aims will be performed: First, sequences both inside and outside the active-site region that affect the ADP-ribosyltransferase reaction will be localized. A model has been developed to help direct these studies, and synthetic peptides and site-specific antibodies will be used as functional probes. Second, the functional role of specific residues in the ADP-ribosyltransferase reaction will be confirmed using site-directed mutagenesis. Third, efforts will be directed at identifying functionally equivalent residues or sequences in different ADP- ribosylating toxins. For this, assays for each step of the ADP- ribosyltransferase reaction will be developed. These assays will then be used to precisely determine the functional role a specific residue plays in the enzymatic reaction. This will be a first step in understanding what defines the common ADP-ribosyltransferase mechanism of these toxins. Fourth, attempts will be made to manipulate toxin reactivity by interchanging functionally equivalent residues between different ADP- ribosylating toxins. With an increased understanding of fine structure/function relationships of ADP-ribosylating toxins will come insight into what determines the ADP- ribosyltransferase reaction and what mechanisms are involved in generating bacterial toxin diversity. This will, in turn, provide direction for the design and genetic construction of toxins with altered specificities and enzymatic activities. If manipulations of bacterial toxin reactivities prove successful, this will set the stage for the long-term objective of these studies which is to develop therapeutic forms of bacterial toxins which specifically target abnormal proteins involved disease processes.